Dipteran insects -- which are responsible for the spreading of malaria, yellow fever, dengue fever, encephalitis and sleeping sickness -- like other insects are protected from their environmental enemies by their tough cuticle. At each molting cycle, the old cuticle is shed and a new cuticle is made to allow growth and development of the insect. Freshly made cuticle is soft and pale, but soon it becomes stiffened by sclerotization reactions involving adduct formation between cuticular proteins, phenols and chitin. The long term objectives is to unravel the structure and mechanism of formation of crosslinks responsible for the stabilization of cuticle and to understand the role of phenoloxidase and related enzymes in sclerotization, wound healing and melanization. The specific aims are: a) to study the biochemistry of quinone methide sclerotization, b) to determine the structure of crosslinks formed in cuticle and c) to study the biochemistry of phenoloxidase-protease cascade in dipterans. To achieve these goals the following studies will be carried out: Studies on quinone methide sclerotization, which was discovered in this laboratory, will be continued using specifically labelled catechols and cuticular enzymes from Sarcophaga bullata and Aedes aegypti. The properties of quinone methide generating enzymes will be studied. Inhibition spectrum by a variety of quinones and catechols will be analyzed for vector control measures. The general occurrence of quinone methide sclerotization in insects will be tested. The structure of adducts formed during quinone tanning and quinone methide tanning will be determined by a combination of chemical, biochemical, and spectroscopic techniques. To assist characterization of natural isolates, synthetic adducts will be made and characterized. Quinone to beta-sclerotization ratio in destabilized mosquito cuticle will be determined. Model sclerotization studies will be continued and noninvasible probes of cuticular structure by ESR will be carried out. These studies on prophenoloxidase-protease- protease inhibitor cascade in S. bullata will be continued and extended to A. aegypti. Using appropriate antibodies, the relationship between soluble hemolymph prophenoloxidase and the cuticular phenoloxidase and the site of synthesis and localization of these components will be determined.